This invention relates to nucleic acid and amino acid sequences of a human SMN-like protein and to the use of these sequences in the diagnosis, treatment, and prevention of neurological, reproductive, and cell proliferative disorders.
Motor neurons directly control muscle activity throughout the body. Motor neuron fibers that extend from the spinal cord to the muscle transmit neural impulses. Motor neuron cell bodies lie within gray matter, the inner core of the spinal cord. They are confined to the anterior horn, one of three distinct functional regions of gray matter. The motor neuron cell bodies receive signals primarily from neurons contained in the other two regions of gray matter. These neurons transmit signals from the brain and other regions of the spinal cord.
Spinal muscular atrophy (SMA) is a fatal neurodegenerative disorder that specifically affects motor neurons of the anterior horn. Extensive loss of these neurons results in progressive muscle weakness and paralysis leading to muscular atrophy. SMA is an autosomal recessive disorder that occurs once in every 6000 live births and has a carrier frequency of 1 in 40. Cystic fibrosis is the only fatal autosomal recessive disorder that occurs with greater frequency. SMA afflicts children, and three types of SMA have been classified based on age of onset and clinical course of the disease. Type I, also called infantile SMA or Werdnig-Hoffman disease, is the most severe form with onset before six months of age and death from respiratory failure by two years of age. Type II, also called chronic childhood SMA or intermediate SMA, presents at around 18 months of age and progresses slowly. Afflicted children cannot walk unaided but survive beyond four years of age. Type III, also called Wohlfart-Kugelberg-Welander disease, is the mildest form with onset ranging from two years of age to adolescence and variable degrees of muscular weakness (Lefebvre et al. (1995) Cell 80:155-165).
SMA is caused by lesions in the survival motor neuron (smn) gene on chromosome 5q13 (Burglen et al. (1996) Genomics 32:479-482). The normal chromosome 5 contains a duplication of the smn locus, resulting in a telomere proximal smn gene (smnT) and a centromere proximal smn gene (smnC). The two genes are nearly identical in nucleotide sequence, and both encode a 294-amino acid protein of 38 kilodaltons. However, the smnC RNA transcript can be alternatively spliced, and the resultant protein is truncated at the C-terminus. The function of this alternative protein product is unknown. Molecular genetic analysis indicates that in over 98% of patients with SMA, smaT is completely or partially deleted. In the remaining 2%, smnT contains point mutations or alterations in splice site consensus sequences. In addition, the severity of the lesion in smnT is correlated with the clinical severity of SMA. These data indicate that smnT, and not smnC, plays a critical role in the determination of SMA (Lefebvre, supra). However, some studies indicate that the activity of smnC may modulate the clinical severity of SMA as previously established by defects in smnT (Coovert et al. (1997) Hum Mol Genet 6:1205-1214). In general, detection of lesions in smnT may provide the basis for definitive prenatal and childhood diagnosis of SMA.
Quantitative western analysis shows that the protein, SMN, is normally expressed at high levels not only in the spinal cord, but also in the kidney, liver, and brain. Intermediate SMN levels are detected in skeletal and cardiac muscle, and low levels are detected in primary fibroblasts and lymphoblasts. The role, if any, for SMN outside of the spinal cord is unclear, as the pathology of SMA is specific to motor neuron muscle control (Coovert, supra). At the cellular level, immunocytochemistry demonstrates that SMN is localized to both the cytoplasm and the nucleus. SMN is diffusely distributed throughout the cytoplasm, while nuclear SMN is concentrated at discrete foci. These foci, called gems, are novel structures that are intimately associated with coiled bodies. Coiled bodies are subnuclear structures involved in RNA processing and metabolism. An in vivo screen for SMN-interacting polypeptides identified fibrillarin, a known component of coiled bodies, and the RGG RNA-binding motif of hnRNP U, a nuclear protein involved in RNA processing. These data suggest that the molecular basis of SMA may involve defects in RNA processing in motor neurons (Liu and Dreyfuss (1996) EMBO J 15:3555-3565).
The mouse homolog of smn has been cloned and localized to chromosome 13 in a region syntenic to that of human chromosome 5q 13. Unlike human smn, mouse smn (msmn) is a single-copy gene, suggesting that duplication of the human locus is a recent evolutionary event. msmn encodes a 288-amino acid protein that shares 82% amino acid identity with human smn. Northern analysis shows that rmsmn RNA is widely expressed in various tissues, including heart, brain, kidney and testis (Viollet et al. (1997) Genomics 40:185-188). Homozygous deletion of msmn is lethal during the morula (16-64 cell) stage of embryogenesis. This phenotype is much more severe than that of SMA in humans, suggesting that differences in gene copy number may influence the severity of the SMA phenotype. In humans, smnC may partially compensate for deletion of smnT to delay disease onset and prolong survival (Schrank et al. (1997) Proc Natl Acad Sci 94:9920-9925).
The discovery of a new human SMN-like protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of neurological, reproductive, and cell proliferative disorders.
The invention is based on the discovery of a human SMN-like protein, HSLP, which shows homology to mouse and human SMN, a protein involved in motor neuron survival. The invention features a substantially purified protein comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides a substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide encoding the protein comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the protein comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the protein comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purified polynucleotide which is complementary to the polynucleotide encoding the protein comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides an isolated and purified polynucleotide having a sequence complementary to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides a polynucleotide fragment comprising nucleotides 712-747 for detecting the presence or expression of an identical endogenous gene.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the protein comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a protein comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the protein comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 under conditions suitable for the expression of the protein; and (b) recovering the protein from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified protein having the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a protein comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist and a purified antagonist of the protein.
The invention also provides a method for treating or preventing a neurological disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified protein having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing a reproductive disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified protein having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing a cell proliferative disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the protein having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for detecting a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide encoding the protein comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the protein comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.